Project Summary Over one billion people worldwide are affected by iron overload, iron deficiency, and states of malnutrition that perturb iron homeostasis. The master regulator of systemic iron metabolism is hepcidin, a hormone that is predominately synthesized and released by the liver. The function of hepcidin is to bind to the only mammalian iron exporter, ferroportin, resulting in ubiquitination, internalization, and degradation of ferroportin. Therefore, in the presence of hepcidin, small amounts of iron are mobilized from stores; in the absence of hepcidin, iron is rapidly mobilized into circulation. Mutations that disrupt the hepcidin/ferroportin signaling axis give rise to iron overload and iron deficiency in mammals, demonstrating that hepcidin and ferroportin are essential for the regulation of systemic iron homeostasis. Our lab has shown that the transcription factor, HIF-2?, is a cellular iron sensor and is the master intestinal regulator of apical and basolateral iron transporters. Moreover, HIF-2? is essential for iron absorption following iron deficiency, the hyperabsorption of iron that leads to tissue iron accumulation during iron overload, and for efficient erythropoiesis. However, it is currently unknown whether the systemic iron regulator, hepcidin, and the intestinal iron regulator, HIF-2?, integrate on the molecular level to maintain organism level iron homeostasis. Using a novel genetic mouse model that allows for tamoxifen-inducible deletion of hepatic hepcidin, our data shows that temporal loss of hepcidin increases intestinal HIF-2? activity and the expression of HIF-2?-specific intestinal iron transporters. Ferroportin is the only known target of hepcidin. To further address this crosstalk, we have also begun to investigate the mechanism by which hepcidin initiates the internalization and degradation of ferroportin, which remains unknown. Our preliminary data shows that, once ubiquitinated and internalized, ferroportin is trafficked to the lysosome independent of canonical macroautophagic machinery. Using a cell based immunoprecipitation approach coupled to mass spectrometry, we identified and confirmed that heat shock 70 kDa protein 8 (HSC70) interacts with ferroportin. HSC70 is the rate limiting cargo protein involved in a process of selective lysosomal degradation that is discrete from macroautophagy, known as chaperone-mediated autophagy. This research proposal will test the hypothesis that rapid activation of intestinal HIF-2? by increased systemic iron demand is mediated by the hepcidin/ferroportin degradation axis in the intestine. This hypothesis will be tested through two interconnected specific aims: (1) Determine the requirement for ferroportin-mediated iron flux in intestinal HIF-2? regulation by hepcidin. (2) Characterize the molecular mechanisms of hepcidin-mediated ferroportin degradation. The proposed studies will unveil the mechanisms by which the liver and the intestine communicate to maintain systemic iron homeostasis, which is essential for the understanding and treatment of iron-related disorders.